Recombinant allergens

ABSTRACT

Novel recombinant allergens are disclosed. The allergens are non-naturally occurring mutants derived from naturally-occurring allergens. The overall α-carbon backbone tertiary structure of the allergens is essentially preserved. Also disclosed are methods for preparing the recombinant allergens as well as the use of the recombinant allergens for the treatment of allergic reactions.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a continuation and claims the benefitof priority under 35 U.S.C. § 120 of Ser. No. 09/270,910, filed Mar. 16,1999, and claims priority under 35 U.S.C. § 119(e) of provisionalapplication No. 60/078,371, filed Mar. 18, 1998. Each of the foregoingapplications is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to novel recombinant allergens,which are non-naturally occurring mutants derived from naturallyoccurring allergens. Further, the invention relates to a method ofpreparing such recombinant allergens as well as to pharmaceuticalcompositions, including vaccines, comprising the recombinant allergens.In further embodiments, the present invention relates to methods ofgenerating immune responses in a subject, vaccination or treatment of asubject as well as processes for preparing the compositions of theinvention.

BACKGROUND OF THE INVENTION

[0003] Genetically predisposed individuals become sensitised (allergic)to antigens originating from a variety of environmental sources, to theallergens of which the individuals are exposed. The allergic reactionoccurs when a previously sensitised individual is re-exposed to the sameor a homologous allergen. Allergic responses range from hay fever,rhinoconductivitis, rhinitis and asthma to systemic anaphylaxis anddeath in response to e.g. bee or hornet sting or insect bite. Thereaction is immediate and can be caused by a variety of atopic allergenssuch as compounds originating from grasses, trees, weeds, insects, food,drugs, chemicals and perfumes.

[0004] However, the responses do not occur when an individual is exposedto an allergen for the first time. The initial adaptive response takestime and does usually not cause any symptoms. But when antibodies and Tcells capable of reacting with the allergen have been produced, anysubsequent exposure may provoke symptoms. Thus, allergic responsesdemonstrate that the immune response itself can cause significantpathological states, which may be life threatening.

[0005] The antibodies involved in atopic allergy belong primarily toimmunoglobulins of the IgE class. IgE binds to specific receptors on thesurface of mast cells and basophils. Following complex formation of aspecific allergen with IgE bound to mast cells, receptor cross-linkingon the cell surface results in signalling through the receptors and thephysiological response of the target cells. Degranulation results in therelease of i.a. histamine, heparin, a chemotactic factor foreosinophilic leukocytes, leukotrienes C4, D4 and E4, which causeprolonged constriction of the bronchial smooth muscle cells. Theresulting effects may be systemic or local in nature.

[0006] The antibody-mediated hypersensitivity reactions can be dividedinto four classes, namely type I, type II, type III and type IV. Type Iallergic reactions is the classic immediate hypersensitivity reactionoccurring within seconds or minutes following antigen exposure. Thesesymptoms are mediated by allergen specific IgE.

[0007] Commonly, allergic reactions are observed as a response toprotein allergens present e.g. in pollens, house dust mites, animal hairand dandruff, venoms, and food products.

[0008] In order to reduce or eliminate allergic reactions, carefullycontrolled and repeated administration of allergy vaccines is commonlyused. Allergy vaccination is traditionally performed by parenteral,intranasal, or sublingual administration in increasing doses over afairly long period of time, and results in desensitisation of thepatient. The exact immunological mechanism is not known, but induceddifferences in the phenotype of allergen specific T cells is thought tobe of particular importance.

[0009] Antibody-Binding Epitopes (B-Cell Epitopes)

[0010] X-ray crystallographic analyses of F_(ab)-antigen complexes hasincreased the understanding of antibody-binding epitopes. According tothis type of analysis antibody-binding epitopes can be defined as asection of the surface of the antigen comprising atoms from 15-25 aminoacid residues, which are within a distance from the atoms of theantibody enabling direct interaction. The affinity of theantigen-antibody interaction can not be predicted from the enthalpycontributed by van der Waals interactions, hydrogen bonds or ionicbonds, alone. The entropy associated with the almost complete expulsionof water molecules from the interface represent an energy contributionsimilar in size. This means that perfect fit between the contours of theinteracting molecules is a principal factor underlying antigen-antibodyhigh affinity interactions.

[0011] Allergy Vaccination

[0012] The concept of vaccination is based on two fundamentalcharacteristics of the immune system, namely specificity and memory.Vaccination will prime the immune system of the recipient, and uponrepeated exposure to similar proteins the immune system will be in aposition to respond more rigorously to the challenge of for example amicrobial infection. Vaccines are mixtures of proteins intended to beused in vaccination for the purpose of generating such a protectiveimmune response in the recipient. The protection will comprise onlycomponents present in the vaccine and homologous antigens.

[0013] Compared to other types of vaccination allergy vaccination iscomplicated by the existence of an ongoing immune response in allergicpatients. This immune response is characterised by the presence ofallergen specific IgE mediating the release of allergic symptoms uponexposure to allergens. Thus, allergy vaccination using allergens fromnatural sources has an inherent risk of side effects being in the utmostconsequence life threatening to the patient.

[0014] Approaches to circumvent this problem may be divided in threecategories. In practise measures from more than one category are oftencombined. First category of measures includes the administration ofseveral small doses over prolonged time to reach a substantialaccumulated dose. Second category of measures includes physicalmodification of the allergens by incorporation of the allergens into gelsubstances such as aluminium hydroxide. Aluminium hydroxide formulationhas an adjuvant effect and a depot effect of slow allergen releasereducing the tissue concentration of active allergen components. Thirdcategory of measures include chemical modification of the allergens forthe purpose of reducing allergenicity, i.e. IgE binding.

[0015] The detailed mechanism behind successful allergy vaccinationremains controversial. It is, however, agreed that T cells play a keyrole in the overall regulation of immune responses. According to currentconsensus the relation between two extremes of T cell phenotypes, Th1and Th2, determine the allergic status of an individual. Uponstimulation with allergen Th1 cells secrete interleukines dominated byinterferon-γ leading to protective immunity and the individual ishealthy. Th2 cells on the other hand secrete predominantly interleukin 4and 5 leading to IgE synthesis and eosinophilia and the individual isallergic. In vitro studies have indicated the possibility of alteringthe responses of allergen specific T cells by challenge with allergenderived peptides containing relevant T cell epitopes. Current approachesto new allergy vaccines are therefore largely based on addressing the Tcells, the aim being to silence the T cells (anergy induction) or toshift the response from the Th2 phenotype to the Th1 phenotype.

[0016] In WO 97/30150 (ref. 1), a population of protein molecules isclaimed, which protein molecules have a distribution of specificmutations in the amino acid sequence as compared to a parent protein.From the description, it appears that the invention is concerned withproducing analogues which are modified as compared to the parentprotein, but which are taken up, digested and presented to T cells inthe same manner as the parent protein (naturally occurring allergens).Thereby, a modified T cell response is obtained. Libraries of modifiedproteins are prepared using a technique denoted PM (ParsimoniousMutagenesis).

[0017] In WO 92/02621 (ref. 2), recombinant DNA molecules are described,which molecules comprise a DNA coding for a polypeptide having at leastone epitope of an allergen of trees of the order Fagales, the allergenbeing selected from Aln g 1, Cor a 1 and Bet v 1. The recombinantmolecules described herein do all have an amino acid sequence or part ofan amino acid sequence that corresponds to the sequence of a naturallyoccurring allergen.

[0018] WO 90/11293 (ref. 3) relates i.a. to isolated allergenic peptidesof ragweed pollen and to modified ragweed pollen peptides. The peptidesdisclosed therein have an amino acid sequence corresponding either tothe sequence of the naturally occurring allergen or to naturallyoccurring isoforms thereof.

[0019] Chemical Modification of Allergens

[0020] Several approaches to chemical modification of allergens havebeen taken. Approaches of the early seventies include chemical couplingof allergens to polymers, and chemical cross-linking of allergens usingformaldehyde, etc., producing the so-called ‘allergoids’. The rationalebehind these approaches was random destruction of IgE binding epitopesby attachment of the chemical ligand thereby reducing IgE-binding whileretaining immunogenicity by the increased molecular weight of thecomplexes. Inherent disadvantages of ‘allergoid’ production are linkedto difficulties in controlling the process of chemical cross-linking anddifficulties in analysis and standardisation of the resulting highmolecular weight complexes. ‘Allergoids’ are currently in clinical useand due to the random destruction of IgE binding epitopes higher dosescan be administered as compared to conventional vaccines, but the safetyand efficacy parameters are not improved over use of conventionalvaccines.

[0021] More recent approaches to chemical modification of allergens aimat a total disruption of the tertiary structure of the allergen thuseliminating IgE binding assuming that the essential therapeutic targetis the allergen specific T cell. Such vaccines contain allergen sequencederived synthetic peptides representing minimal T cells epitopes, longerpeptides representing linked T cells epitopes, longer allergen sequencederived synthetic peptides representing regions of immunodominant T cellepitopes, or allergen molecules cut in two halves by recombinanttechnique. Another approach based on this rationale has been theproposal of the use of “low IgE binding” recombinant isoforms. In recentyears it has become clear that natural allergens are heterogeneouscontaining isoallergens and variants having up to approximately 25% oftheir amino acids substituted. Some recombinant isoallergens have beenfound to be less efficient in IgE binding possibly due to irreversibledenaturation and hence total disruption of tertiary structure.

[0022] In Vitro Mutagenesis and Allergy Vaccination

[0023] Attempts to reduce allergenicity by in vitro site directedmutagenesis have been performed using several allergens including Der f2 (Takai et al, ref. 4), Der p 2 (Smith et al, ref. 5), a 39 kDaDermatophagoides farinae allergen (Aki et al, ref. 6), bee venomphospholipase A2 (Förster et al, ref. 7), Ara h 1 (Burks et al, ref. 8),Ara h 2 (Stanley et al, ref. 9), Bet v 1 (Ferreira et al, ref. 10 and11), birch profilin (Wiedemann et al, ref. 12), and Ory s 1 (Alvarez etal, ref. 13).

[0024] The rationale behind these approaches, again, is addressingallergen specific T cells while at the same time reducing the risk ofIgE mediated side effects by reduction or elimination of IgE binding bydisruption of the tertiary structure of the recombinant mutant allergen.The rationale behind these approaches does not include the concept ofdominant IgE binding epitopes and it does not include the concept ofinitiating a new protective immune response which also involves B-cellsand antibody generation.

[0025] The article by Ferreira et al (ref. 11) describes the use of sitedirected mutagenesis for the purpose of reducing IgE binding. Althoughthe three-dimensional structure of Bet v 1 is mentioned in the articlethe authors do not use the structure for prediction of surface exposedamino acid residues for mutation, half of which have a low degree ofsolvent exposure. Rather they use a method developed for prediction offunctional residues in proteins different from the concept of structurebased identification of conserved surface areas described here. Althoughthe authors do discuss conservation of α-carbon backbone tertiarystructure this concept is not a part of the therapeutic strategy butmerely included to assess in vitro IgE binding. Furthermore, theevidence presented is not adequate since normalisation of CD-spectraprevents the evaluation of denaturation of a proportion of the sample,which is a common problem. The therapeutic strategy described aim atinducing tolerance in allergen specific T cells and initiation of a newimmune response is not mentioned.

[0026] The article by Wiedemann et al. (ref. 12) describes the use ofsite directed mutagenesis and peptide synthesis for the purpose ofmonoclonal antibody epitope characterisation. The authors have knowledgeof the tertiary structure of the antigen and they use this knowledge toselect a surface exposed amino acid for mutation. The algorithm used canbe said to be opposite to the one described by the present inventorssince an amino acid differing from homologous sequences is selected. Thestudy demonstrates that substitution of a surface exposed amino acid hasthe capacity to modify the binding characteristics of a monoclonalantibody, which is not surprising considering common knowledge. Theexperiments described are not designed to assess modulation in thebinding of polyclonal antibodies such as allergic patients' serum IgE.One of the experiments contained do apply serum IgE and although thisexperiment is not suitable for quantitative assessment, IgE binding doesnot seem to be affected by the mutations performed.

[0027] The article by Smith et al. (ref. 5) describes the use of sitedirected mutagenesis for the purpose of monoclonal antibody epitopemapping and reduction of IgE binding. The authors have no knowledge ofthe tertiary structure and make no attempt to assess the conservation ofα-carbon backbone tertiary structure. The algorithm used does not ensurethat amino acids selected for mutation are actually exposed to themolecular surface. Only one of the mutants described lead to asubstantial reduction in IgE binding. This mutant is deficient inbinding of all antibodies tested indicating that the tertiary structureis disrupted. The authors do not define a therapeutic strategy andinitiation of a new immune response is not mentioned.

[0028] The article by Colombo et al. (ref. 14) describes the study of anIgE binding epitope by use of site directed mutagenesis and peptidesynthesis. The authors use a three dimensional computer model structurebased on the crystal structure of a homologous protein to illustrate thepresence of the epitope on the molecular surface. The further presenceof an epitope on a different allergen showing primary structure homologyis addressed using synthetic peptides representing the epitope. Thetherapeutic strategy is based on treatment using this synthetic peptiderepresenting a monovalent IgE binding epitope. Conserved surface areasbetween homologous allergens as well as the therapeutic concept ofinitiating a new protective immune response are not mentioned.

[0029] The article by Spangfort et al. (ref. 15) describes thethree-dimensional structure and conserved surface exposed patches of themajor birch allergen. The article does not mention major IgE bindingepitopes nor site directed mutagenesis, neither is therapeuticapplication addressed.

[0030] In none of the studies described above is IgE binding reduced bysubstitution of surface exposed amino acids while conserving α-carbonbackbone tertiary structure. The rationale behind above-mentionedapproaches does not include the concept of dominant IgE binding epitopesand it does not include the therapeutic concept of initiating a newprotective immune response.

BRIEF DESCRIPTION OF THE FIGURES

[0031]FIG. 1 shows mutant-specific oligonucleotide primers used for Betv 1 mutant number 1. Mutated nucleotides are underlined (SEQ ID NOS:1-4).

[0032]FIG. 2 shows two generally applicable primers (denoted “all-sense”and “all non-sense”), which were synthesised and used for all mutants(SEQ ID NOS: 5-22).

[0033]FIG. 3 shows an overview of all Bet v 1 mutations (SEQ ID NOS:36-37).

[0034]FIG. 4 shows the inhibition of the binding of biotinylatedrecombinant Bet v 1 to serum IgE from a pool of allergic patients bynon-biotinylated Bet v 1 and by Bet v 1 Glu45Ser mutant.

[0035]FIG. 5 shows the inhibition of the binding of biotinylatedrecombinant Bet v 1 to serum IgE from a pool of allergic patients bynon-biotinylated Bet v 1 and by Bet v 1 mutant Asn28Thr+Lys32Gln.

[0036]FIG. 6 shows the inhibition of the binding of biotinylatedrecombinant Bet v 1 to serum IgE from a pool of allergic patients bynon-biotinylated Bet v 1 and by Bet v 1 Pro108Gly mutant.

[0037]FIG. 7 shows the inhibition of the binding of biotinylatedrecombinant Bet v 1 to serum IgE from a pool of allergic patients bynon-biotinylated Bet v 1 and by Bet v 1 Glu60Ser mutant.

[0038]FIG. 8 shows the CD spectra of recombinant and Triple-patchmutant, recorded at close to equal concentrations.

[0039]FIG. 9 shows the inhibition of the binding of biotinylatedrecombinant Bet v 1 to serum IgE from a pool of allergic patients bynon-biotinylated Bet v 1 and by Bet v 1 Triple-patch mutant.

[0040]FIG. 10 shows solvent accessibility of individually alignedantigen 5 residues and alignment of Vespula antigen 5 sequences (leftpanel). On the right panel of FIG. 10 is shown the molecular surface ofantigen 5 with conserved areas among Vespula antigen 5:s.

[0041]FIG. 11 shows the sequence of the primer corresponding to theamino terminus of Ves v 5 derived from the sense strand (SEQ ID NOS:23-30). The sequence of the downstream primer is derived from thenon-sense strand.

[0042]FIG. 12 shows two generally applicable primers (denoted “allsense” and “all non-sense”, which were synthesised and used for allmutants (SEQ ID NOS: 31-35).

[0043]FIG. 13 shows an overview of all Ves v 5 mutations (SEQ ID NOS:38-39).

[0044]FIG. 14 shows the inhibition of the binding of biotinylatedrecombinant Ves v 5 to serum IgE from a pool of allergic patients bynon-biotinylated Ves v 5 and by Ves v 5 Lys72Ala mutant.

OBJECT OF THE INVENTION

[0045] Rationale Behind the Present Invention

[0046] The current invention is based on a unique rationale. Accordingto this rationale the mechanism of successful allergy vaccination is notan alteration of the ongoing Th2-type immune response, but rather aparallel initiation of a new Th1-type immune response involving tertiaryepitope recognition by B-cells and antibody formation. This model issupported by the observation that levels of specific IgE are unaffectedby successful vaccination treatment, and that successful treatment isoften accompanied by a substantial rise in allergen specific IgG4. Inaddition, studies of nasal biopsies before and after allergen challengedo not show a reduction in T cells with the Th2-like phenotype, butrather an increase in Th1-like T cells are observed. When the vaccine(or pharmaceutical compositions) is administered through another routethan the airways, it is hypothesised, that the new Th1-like immuneresponse evolves in a location physically separated from the ongoing Th2response thereby enabling the two responses to exist in parallel.

[0047] Another important aspect of the rationale behind the currentinvention is the assertion of the existence of dominant IgE bindingepitopes. It is proposed that these dominant IgE binding epitopes areconstituted by tertiary structure dependent coherent surface areas largeenough to accommodate antibody binding and conserved among isoallergens,variants, and/or homologous allergens from related species. Theexistence of cross-reactive IgE capable of binding similar epitopes onhomologous allergens is supported by the clinical observation thatallergic patients often react to several closely related species, e.g.alder, birch (SEQ ID NOS: 36-37), and hazel, multiple grass species, orseveral species of the house dust mite genus Dermatophagoides. It isfurthermore supported by laboratory experiments demonstrating IgEcross-reactivity between homologous allergens from related species andthe capacity of one allergen to inhibit the binding of IgE to homologousallergens (Ipsen et al. 1992, ref. 16). It is well known that exposureand immune responses are related in a dose dependent fashion. Based onthe combination of these observations it is hypothesised that conservedsurface areas are exposed to the immune system in higher doses thannon-conserved surface areas resulting in the generation of IgEantibodies with higher affinities, hence the term ‘dominant IgE bindingepitopes’.

[0048] According to this rationale it is essential that the allergen hasan α-carbon backbone tertiary structure which essentially is the same asthat of the natural allergen, thus ensuring conservation of the surfacetopology of areas surrounding conserved patches representing targets formutagenesis aimed at reducing IgE binding. By fulfilling these criteriathe allergen has the potential to be administered in relatively higherdoses improving its efficacy in generating a protective immune responsewithout compromising safety.

SUMMARY OF THE INVENTION

[0049] The present invention relates to the introduction of artificialamino acid substitutions into defined critical positions while retainingthe α-carbon backbone tertiary structure of the allergen.

[0050] The invention provides a recombinant allergen, which is anon-naturally occurring mutant derived from a naturally occurringallergen, wherein at least one surface-exposed, conserved amino acidresidue of a B cell epitope is substituted by another residue which doesnot occur in the same position in the amino acid sequence of any knownhomologous protein within the taxonomic order from which said naturallyoccurring allergen originates, said mutant allergen having essentiallythe same α-carbon backbone tertiary structure as said naturallyoccurring allergen, and the specific IgE binding to the mutated allergenbeing reduced as compared to the binding to said naturally occurringallergen.

[0051] Such recombinant allergen is obtainable by

[0052] a) identifying amino acid residues in a naturally occurringallergen which are conserved with more than 70% identity in all knownhomologous proteins within the taxonomic order from which said naturallyoccurring allergen originates;

[0053] b) defining at least one patch of conserved amino acid residuesbeing coherently connected over at least 400 Å² of the surface of thethree-dimensional of the allergen molecule as defined by having asolvent accessibility of at least 20%, said at least one patchcomprising at least one B cell epitope; and

[0054] c) substituting at least one amino acid residue in said at leastone patch by another amino acid being non-conservative in the particularposition while essentially preserving the overall α-carbon backbonetertiary structure of the allergen molecule.

[0055] Specific IgE binding to the mutated allergen is preferablyreduced by at least 5%, preferably at least 10% in comparison tonaturally-occurring isoallergens or similar recombinant proteins in animmuno assay with sera from scource-specific IgE reactive allergicpatients or pools thereof.

[0056] Recombinant allergens according to the invention may suitably bederived from inhalation allergens originating i.a. from trees, grasses,herbs, fungi, house dust mites, cockroaches and animal hair anddandruff. Important pollen allergens from trees, grasses and herbs aresuch originating from the taxonomic orders of Fagales, Oleales andPinales including i.a. birch (Betula), alder (Alnus), hazel (Corylus),hornbeam (Carpinus) and olive (Olea), the order of Poales including i.a.grasses of the genera Lolium, Phelum, Poa, Cynodon, Dactylis and Secale,the orders of Asterales and Urticales including i.a. herbs of the generaAmbrosia and Artemisia. Important inhalation allergens from fungi arei.a. such originating from the genera Alternaria and Cladosporium. Otherimportant inhalation allergens are those from house dust mites of thegenus Dermatophagoides, those from cockroaches and those from mammalssuch as cat, dog and horse. Further, recombinant allergens according tothe invention may be derived from venom allergens including suchoriginating from stinging or biting insects such as those from thetaxonomic order of Hymenoptera including bees (superfamily Apidae),wasps (superfamily Vespidea), and ants (superfamily Formicoidae).

[0057] Specific allergen components include e.g. Bet v 1 (B. verrucosa,birch), Aln g 1 (Alnus glutinosa, alder), Cor a 1 (Corylus avelana,hazel) and Car b 1 (Carpinus betulus, hornbeam) of the Fagales order.Others are Cry j 1 (Pinales), Amb a 1 and 2, Art v 1 (Asterales), Par j1 (Urticales), Ole e 1 (Oleales), Ave e 1, Cyn d 1, Dac g 1, Fes p 1,Hol l 1, Lol p 1 and 5, Pas n 1, Phl p 1 and 5, Poa p 1, 2 and 5, Sec c1 and 5, and Sor h 1 (various grass pollens), Alt a 1 and Cla h 1(fungi), Der f 1 and 2, Der p 1 and 2 (house dust mites, D. farinae andD. pteronyssinus, respectively), Bla g 1 and 2, Per a 1 (cockroaches,Blatella germanica and Periplaneta americana, respectively), Fel d 1(cat), Can f 1 (dog), Equ c 1, 2 and 3 (horse), Apis m 1 and 2(honeybee), Ves g 1, 2 and 5, Pol a 1, 2 and 5 (all wasps) and Sol i 1,2, 3 and 4 (fire ant).

[0058] In one embodiment, the recombinant allergen is derived from Bet v1 (SEQ ID NOS: 36-37). Examples of substitutions are Thr10Pro, Asp25Gly,(Asn28Thr+Lys32Gln), Glu45Ser, Asn47Ser, Lys55Asn, Thr77Ala, Pro108Glyand (Asn28Thr, Lys32Gln, Glu45Ser, Pro108Gly. As apparent, therecombinant allergens may have one or more substitutions.

[0059] In another embodiment, the recombinant allergen is derived from avenom allergen from the taxonomic order of Vespidae, Apidae andFormicoidae.

[0060] In a further embodiment, the recombinant allergen is derived fromVes v 5 (SEQ ID NOS: 38-39). Examples of substitutions are Lys72Ala andTyr96Ala. As apparent, the recombinant allergens may have one or moresubstitutions.

[0061] The present invention also provides a method of preparing arecombinant allergen as defined herein, comprising

[0062] a) identifying amino acid residues in a naturally occurringallergen which are conserved with more than 70% identity in all knownhomologous proteins within the taxonomic order from which said naturallyoccurring allergen originates;

[0063] b) defining at least one patch of conserved amino acid residuesbeing coherently connected over at least 400 Å² of the surface of thethree-dimensional structure of the allergen molecule as defined byhaving a solvent accessibility of at least 20%, said at least one patchcomprising at least one B cell epitope, and

[0064] c) substituting at least one amino acid residue in said at leastone patch by another amino acid being non-conservative in the particularposition while essentially preserving the overall α-carbon backbonetertiary structure of the allergen molecule.

[0065] In this method the best results are obtained by ranking the aminoacid residues of said at least one patch with respect to solventaccessibility and substituting one or more amino acids among the moresolvent accessible ones.

[0066] Generally, in the method according to the invention thesubstitution of one or more amino acid residues in said B cell epitopeor said at least one patch is carried out by site-directed mutagenesis.

[0067] Conservation of α-carbon backbone tertiary structure is bestdetermined by obtaining identical structures by x-ray crystallography orNMR before and after mutagenesis. In absence of structural datadescribing the mutant indistinguishable CD-spectra or immunochemicaldata, e.g. antibody reactivity, may render conservation of α-carbonbackbone tertiary structure probable, if compared to the data obtainedby analysis of a structurally determined molecule.

[0068] Further, the present invention provides a pharmaceuticalcomposition comprising a recombinant allergen as defined herein incombination with a pharmaceutically acceptable carrier and/or excipient,and optionally an adjuvant.

[0069] Such pharmaceutical composition may be in the form of a vaccineagainst allergic reactions elicited by a naturally occurring allergen inpatients suffering from allergy.

[0070] In a further aspect, the present invention relates to a method ofgenerating an immune response in a subject, which method comprisesadministering to the subject at least one recombinant allergen asdefined herein, or a pharmaceutical composition comprising at least onerecombinant allergen as defined herein.

[0071] The pharmaceutical composition of the invention can be preparedby a process comprising mixing at least one recombinant allergen asdefined herein with pharmaceutically acceptable substances and/orexcipients.

[0072] In a particular embodiment, the present invention concerns thevaccination or treatment of a subject, which vaccination of treatmentcomprises administering to the subject at least one recombinant allergenas defined herein or a pharmaceutical composition as defined herein.

[0073] The pharmaceutical compositions of the invention are obtainableby the process defined above.

[0074] In another embodiment, the recombinant allergens of the inventionare suitable for use in a method for the treatment, prevention oralleviation of allergic reactions, such method comprising administeringto a subject a recombinant allergen as defined herein or apharmaceutical composition as defined herein.

DETAILED DESCRIPTION OF THE INVENTION

[0075] Criteria for Substitution

[0076] For molecules for which the tertiary structure has beendetermined (e.g. by x-ray crystallography, or NMR electron microscopy),the mutant carrying the substituted amino acid(s) should preferablyfulfil the following criteria:

[0077] 1. The overall α-carbon backbone tertiary structure of themolecule should be conserved. Conserved is defined as an average rootmean square deviation of the atomic coordinates comparing the structuresbelow 2 Å. This is important for two reasons: a) It is anticipated thatthe entire surface of the natural allergen constitutes an overlappingcontinuum of potential antibody-binding epitopes. The majority of thesurface of the molecule is not affected by the substitution(s), and thusretain its antibody-binding properties, which is important for thegeneration of new protective antibody specificities being directed atepitopes present also on the natural allergen. b) Stability, bothconcerning shelf-life and upon injection into body fluids.

[0078] 2. The amino acid(s) to be substituted should be located at thesurface, and thus be accessible for antibody-binding. Amino acidslocated on the surface are defined as amino acids in thethree-dimensional structure having a solvent (water) accessibility of atleast 20%, suitably 20-80%, more suitably 30-80%. Solvent accessibilityis defined as the area of the molecule accessible to a sphere with aradius comparable to a solvent (water, r=1.4 Å) molecule.

[0079] 3. The substituted amino acid(s) should be located in conservedpatches larger than 400 Å². Conserved patches are defined as coherentlyconnected areas of surface exposed conserved amino acid residues andbackbone. Conserved amino acid residues are defined by sequencealignment of all known (deduced) amino acid sequences of homologuesproteins within the taxonomical order. Amino acid positions havingidentical amino acid residues in more than 90% of the sequences areconsidered conserved. Conserved patches are expected to contain epitopesto which the IgE of the majority of patients is directed.

[0080] 4. Within the conserved patches amino acids for mutagenesisshould preferentially be selected among the most solvent (water)accessible ones located preferably near the centre of the conservedpatch.

[0081] Preferentially, a polar amino acid residue is substituted byanother polar residue, and a non-polar amino acid residue is substitutedby another non-polar residue.

[0082] Preparation of vaccines is generally well-known in the art.Vaccines are typically prepared as injectables either as liquidsolutions or suspensions. Such vaccine may also be emulsified orformulated so as to enable nasal administration. The immunogeniccomponent in question (the recombinant allergen as defined herein) maysuitably be mixed with excipients which are pharmaceutically acceptableand further compatible with the active ingredient. Examples of suitableexcipients are water, saline, dextrose, glycerol, ethanol and the likeas well as combinations thereof. The vaccine may additionally containother substances such as wetting agents, emulsifying agents, bufferingagents or adjuvants enhancing the effectiveness of the vaccine.

[0083] Vaccines are most frequently administered parenterally bysubcutaneous or intramuscular injection. Formulations which are suitablefor administration by another route include oral formulations andsuppositories. Vaccines for oral administration may suitably beformulated with excipients normally employed for such formulations, e.g.pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate and the like. Thecomposition can be formulated as solutions, suspensions; emulsions,tablets, pills, capsules, sustained release formulations, aerosols,powders, or granulates.

[0084] The vaccines are administered in a way so as to be compatiblewith the dosage formulation and in such amount as will betherapeutically effective and immunogenic. The quantity of activecomponent contained within the vaccine depends on the subject to betreated, i.a. the capability of the subject's immune system to respondto the treatment, the route of administration and the age and weight ofthe subject. Suitable dosage ranges can vary within the range from about0.0001 μg to 1000 μg.

[0085] As mentioned above, an increased effect may be obtained by addingadjuvants to the formulation. Examples of such adjuvants are aluminumhydroxide and phosphate (alum) as a 0.05 to 0.1 percent solution inphosphate buffered saline, synthetic polymers of sugars used as 0.25percent solution. Mixture with bacterial cells such as C. parvum,endotoxins or lipopolysaccharide components of gram-negative bacteria,emulsion in physiologically acceptable oil vehicles such as mannidemonoaleate (Aracel A) or emulsion with 20 percent solution of aperfluorocarbon (e.g. Fluosol-DA) used as a block substitute may also beemployed. Other adjuvants such as Freund's complete and incompleteadjuvants as well as QuilA and RIBI may also be used.

[0086] Most often, multiple administrations of the vaccine will benecessary to ensure an effect. Frequently, the vaccine is administeredas an initial administration followed by subsequent inoculations orother administrations. The number of vaccinations will typically be inthe range of from 1 to 50, usually not exceeding 35 vaccinations.Vaccination will normally be performed from biweekly to bimonthly for aperiod of 3 months to 5 years. This is contemplated to give desiredlevel of prophylactic or therapeutic effect.

[0087] The recombinant allergen may be used as a pharmaceuticalpreparation, which is suitable for providing a certain protectionagainst allergic responses during the period of the year where symptomsoccur (prophylaxis). Usually, the treatment will have to be repeatedevery year to maintain the protective effect. Preparations formulatedfor nasal application are particular suited for this purpose.

[0088] The present invention is further illustrated by the followingnon-limiting examples.

EXAMPLES Example 1

[0089] Identification of Common Epitopes within Fagales Pollen Allergens

[0090] The major birch pollen allergen Bet v 1 (SEQ ID NO: 37) showsabout 90% amino acid sequence identity with major allergens from pollensof taxonomically related trees, i.e Fagales (for instance hazel andhornbeam) and birch pollen allergic patients often show clinicalsymptoms of allergic cross-reactivity towards these Bet v 1 homologousproteins.

[0091] Bet v 1 (SEQ ID NO: 37) also shows about 50-60% sequence identitywith allergic proteins present in certain fruits (for instance apple andcherry) and vegetables (for instance celery and carrot) and there areclinical evidence for allergic cross-reactivity between Bet v 1 (SEQ IDNO: 37) and these food related proteins.

[0092] In addition, Bet v 1 shares significant sequence identity(20-40%) with a group of plant proteins called pathogenesis-relatedproteins (PR-10), however there are no reports of allergiccross-reactivity towards these PR-10 proteins.

[0093] Molecular modelling suggests that the structures of Fagales andfood allergens and PR-10 proteins are close to be identical with the Betv 1 (SEQ ID NO: 37) structure.

[0094] The structural basis for allergic Bet v 1 (SEQ ID NO: 37)cross-reactivity was reported in (Gajhede et al 1996, ref. 17) wherethree patches on the molecular surface of Bet v 1 (SEQ ID NO: 37) couldbe identified to be common for the known major tree pollen allergens.Thus, any IgE recognising these patches on Bet v 1 (SEQ ID NO: 37) wouldbe able to cross-react and bind to other Fagales major pollen allergensand give rise to allergic symptoms. The identification of these commonpatches was performed after alignment of all known amino acid sequencesof the major tree pollen allergens in combination with an analysis ofthe molecular surface of Bet v 1 (SEQ ID NO: 37) revealed by theα-carbon backbone tertiary structure reported in ref. 17. In addition,the patches were defined to have a certain minimum size (>400 Å²) basedon the area covered by an antibody upon binding.

[0095] Selection of Amino Acid Residues for Site-Directed Mutagenesis

[0096] Amino acid residues for site-directed mutagenesis were selectedamong residues present in Bet v 1 (SEQ ID NO: 37) specific areas and thecommon patches since modifications of these is expected to affect thebinding of serum IgE from the majority of patients showing clinical treepollen allergic cross-reactivity.

[0097] The relative orientation and percentage of solvent-exposure ofeach amino acid residue within respective patch was calculated based ontheir atomic coordinates. Residues having a low degree of solventexposure (<20%) were not regarded relevant for mutagenesis due to thepossible disruption of the structure or lack of antibody interaction.The remaining residues were ranked according to their degree ofsolvent-exposure.

[0098] Sequence Alignment

[0099] Sequences homologous to the query sequence (Bet v 1 No. 2801, WHOIUIS Nomenclature Subcommittee on Allergens (SEQ ID NOS: 36-37) werederived from GenBank and EMBL sequence databases by a BLAST search(Altschul et al., ref. 18). All sequences with BLAST reportedprobabilities less than 0.1 were taken into consideration and one listwere constructed containing a non-redundant list of homologoussequences. These were aligned by CLUSTAL W (Higgins et al., ref. 19) andthe percentage identity were calculated for each position in thesequence considering the complete list or taxonomically related speciesonly. A total of 122 sequences were homologous to Bet v 1 No. 2801 (SEQID NOS: 36-37) of which 57 sequences originates from taxonomicallyrelated species.

[0100] Cloning of the gene encoding Bet v 1 (SEQ ID NO: 36)

[0101] RNA was prepared from Betula verrucosa pollen (Allergon, Sweden)by phenol extraction and LiCl precipitation. Oligo(dT)-celluloseaffinity chromatography was performed batch-wise in Eppendorph tubes,and double-stranded cDNA was synthesised using a commercially availablekit (Amersham). DNA encoding Bet v 1 (SEQ ID NO: 36) was amplified byPCR and cloned. In brief, PCR was performed using cDNA as template, andprimers designed to match the sequence of the cDNA in positionscorresponding to the amino terminus of Bet v 1 (SEQ ID NO: 37) and the3′-untranslated region, respectively. The primers were extended in the5′-ends to accommodate restriction sites (NcoI and HindIII) fordirectional cloning into pKK233-2.

[0102] Subcloning into pMAL-c

[0103] The gene encoding Bet v 1 (SEQ ID NO: 36) was subsequentlysubcloned into the maltose binding protein fusion vector pMAL-c (NewEngland Biolabs). The gene was amplified by PCR and subcloned in framewith malE to generate maltose binding protein (MBP)-Bet v 1 (SEQ ID NO:37) protein fusion operons in which MBP and Bet v 1 were separated by afactor X_(a) protease clevage site positioned to restore the authenticaminoterminal sequence of Bet v 1 (SEQ ID NO: 37) upon cleavage, asdescribed in ref. 15. In brief, PCR was performed using pKK233-3 withBet v 1 (SEQ ID NO: 36) inserted as template and primers correspondingto the amino- and carboxyterminus of the protein, respectively. Thepromoter proximal primer was extended in the 5′-end to accommodate 4codons encoding an in frame factor X_(a) protease cleavage site. Bothprimers were furthermore extended in the 5′-ends to accommodaterestriction sites (KpnI) for cloning. The Bet v 1 encoding genes weresubcloned using 20 cycles of PCR to reduce the frequency of PCRartefacts.

[0104] In Vitro Mutagenesis

[0105] In vitro mutagenesis was performed by PCR using recombinantpMAL-c with Bet v 1 inserted as template. Each mutant Bet v 1 gene wasgenerated by 3 PCR reactions using 4 primers.

[0106] Two mutation-specific oligonucleotide primers were synthesisedaccommodating each mutation, one for each DNA strand, see FIGS. 1 and 2,Using the mutated nucleotide(s) as starting point both primers wereextended 7 nucleotides in the 5′-end and 15 nucleotides in the 3′-end.The extending nucleotides were identical in sequence to the Bet v 1 genein the actual region.

[0107] Two generally applicable primers (denoted “all-sense” and “allnon-sense” in FIG. 2) were furthermore synthesised and used for allmutants. These primers were 15 nucleotides in length and correspond insequence to regions of the pMAL-c vector approximately 1 kilobaseupstream and downstream from the Bet v 1. The sequence of the upstreamprimer is derived from the sense strand and the sequence of thedownstream primer is derived from the non-sense strand, see FIG. 2.

[0108] Two independent PCR reactions were performed essentiallyaccording to standard procedures (Saiki et al 1988, ref. 20) with theexception that only 20 temperature cycles were performed in order toreduce the frequency of PCR artefacts. Each PCR reaction used pMAL-cwith Bet v 1 inserted as template and one mutation-specific and onegenerally applicable primer in meaningful combinations.

[0109] Introduction of the four amino acid substitutions (Asn28Thr,Lys32Gln, Glu45Ser, Pro108Gly) in the Triple-patch mutant were performedlike described above in a step by step process (SEQ ID NO: 37). Firstthe Glu45Ser mutation then the Pro108Gly mutation and last the Asn28Thr,Lys32Gln mutations were introduced using pMAL-c with inserted Bet v 1No. 2801, Bet v 1 (Glu45Ser), Bet v 1 (Glu45Ser, Pro108Gly) astemplates, respectively (SEQ ID NO: 37).

[0110] The PCR products were purified by agarose gel electrophoresis andelectro-elution followed by ethanol precipitation. A third PCR reactionwas performed using the combined PCR products from the first two PCRreactions as template and both generally applicable primers. Again, 20cycles of standard PCR were used. The PCR product was purified byagarose gel electrophoresis and electro-elution followed by ethanolprecipitation, cut with restriction enzymes (BsiWI/EcoRI), and ligateddirectionally into pMAL-c with Bet v 1 (SEQ ID NO: 36) insertedrestricted with the same enzymes.

[0111]FIG. 3 shows an overview of all 9 Bet v 1 mutations, which are asfollows (SEQ ID NOS: 36-37):

[0112] Thr10Pro, Asp25Gly, Asn28Thr+Lys32Gln, Glu45Ser, Asn47Ser,Lys55Asn, Glu60Ser (non-patch), Thr77Ala and Pro108Gly (SEQ ID NO: 37).An additional four mutant with four mutations was also prepared(Asn28Thr, Lys32Gln, Glu45Ser, Pro108Gly) (SEQ ID NO: 37). Of these,five were selected for further testing: Asn28Thr+Lys32Gln, Glu45Ser,Glu60Ser, Pro108Gly and the Triple-patch mutant Asn28Thr, Lys32Gln,Glu45Ser, Pro108Gly (SEQ ID NO: 37).

[0113] Nucleotide Sequencing

[0114] Determination of the nucleotide sequence of the Bet v 1 encodinggene (SEQ ID NO: 36) was performed before and after subcloning, andfollowing in vitro mutagenesis, respectively.

[0115] Plasmid DNA's from 10 ml of bacterial culture grown to saturationovernight in LB medium supplemented with 0.1 g/l ampicillin werepurified on Qiagen-tip 20 columns and sequenced using the Sequenaseversion 2.0 DNA sequencing kit (USB) following the recommendations ofthe suppliers.

[0116] Expression and Purification of Recombinant Bet v 1 and Mutants

[0117] Recombinant Bet v 1 (Bet v 1 No. 2801 and mutants (SEQ ID NO: 37)were over-expressed in Escherichia coli DH 5a fused to maltose-bindingprotein and purified as described in ref. 15. Briefly, recombinant E.coli cells were grown at 37° C. to an optical density of 1.0 at 436 nm,whereupon expression of the Bet v 1 fusion protein was induced byaddition of IPTG. Cells were harvested by centrifugation 3 hourspost-induction, re-suspended in lysis buffer and broken by sonication.After sonication and additional centrifugation, recombinant fusionprotein was isolated by amylose affinity chromatography and subsequentlycleaved by incubation with Factor Xa (ref. 15). After F Xa cleavage,recombinant Bet v 1 (SEQ ID NO: 37) was isolated by gelfiltration and iffound necessary, subjected to another round of amylose affinitychromatography in order to remove trace amounts of maltose-bindingprotein.

[0118] Purified recombinant Bet v 1 (SEQ ID NO: 37) was concentrated byultrafiltration to about 5 mg/ml and stored at 4° C. The final yields ofthe purified recombinant Bet v 1 (SEQ ID NO: 37) preparations werebetween 2-5 mg per litre E. coli cell culture.

[0119] The purified recombinant Bet v 1 (SEQ ID NO: 37) preparationsappeared as single bands after silver-stained SDS-polyacrylamideelectrophoresis with an apparent molecular weight of 17.5 kDa.N-terminal sequencing showed the expected sequences as derived from thecDNA nucleotide sequences and quantitative amino acid analysis showedthe expected amino acid compositions.

[0120] We have previously shown (ref. 15) that recombinant Bet v 1 No.2801 (SEQ ID NO: 37) is immunochemically indistinguishable fromnaturally occurring Bet v 1.

[0121] Immunoelectrophoresis Using Rabbit Polyclonal Antibodies

[0122] The seven mutant Bet v 1 (SEQ ID NO: 37) were produced asrecombinant Bet v 1 proteins (SEQ ID NO: 37) and purified as describedabove and tested for their reactivity towards polyclonal rabbitantibodies raised against Bet v 1 (SEQ ID NO: 37) isolated from birchpollen. When analysed by immunoelectrophoresis (rocket-lineimmunoelectrophoresis) under native conditions, the rabbit antibodieswere able to precipitate all mutants, indicating that the mutants hadconserved α-carbon backbone tertiary structure.

[0123] These results suggested that non-naturally occurringsubstitutions introduced on the molecular surface of Bet v 1 (SEQ ID NO:37) can reduce a polyclonal antibody response raised against naturallyoccurring Bet v 1 (SEQ ID NO: 37) without distortion of the overallα-carbon backbone tertiary allergen structure. In order to analyse theeffect on human polyclonal IgE-response, the mutants Glu45Ser,Pro108Gly, Asn28Thr+Lys32Gln and Glu60Ser were selected for furtheranalysis (SEQ ID NO: 37).

[0124] Bet v 1 Glu45Ser Mutant (SEQ ID NO: 37)

[0125] Glutamic acid in position 45 show a high degree ofsolvent-exposure (40%) and is located in a molecular surface patchcommon for Fagales allergens (patch I). A serine residue was found tooccupy position 45 in some of the Bet v 1 (SEQ ID NO: 37) homologousPR-10 proteins arguing for that glutamic acid can be replaced by serinewithout distortion of the α-carbon backbone tertiary structure. Inaddition, as none of the known Fagales allergen sequences have serine inposition 45, the substitution of glutamic acid with serine gives rise toa non-naturally occurring Bet v 1 molecule (SEQ ID NO: 37).

[0126] T Cell Proliferation Assay Using Recombinant Glu45Ser Bet v 1Mutant (SEQ ID NO: 37)

[0127] The analysis was carried out as described in Spangfort et al1996a. It was found that recombinant Bet v 1 Glu45Ser mutant was able toinduce proliferation in T cell lines from three different birch pollenallergic patients with stimulation indices similar to recombinant andnaturally occurring.

[0128] Crystallisation and Structural Determination of RecombinantGlu45Ser Bet v 1 (SEQ ID NO: 37)

[0129] Crystals of recombinant Glu45Ser Bet v 1 (SEQ ID NO: 37) weregrown by vapour diffusion at 25° C., essentially as described in(Spangfort et al 1996b, ref. 21). Glu45Ser Bet v 1 (SEQ ID NO: 37), at aconcentration of 5 mg/ml, was mixed with an equal volume of 2.0 Mammonium sulphate, 0.1 M sodium citrate, 1% (v/v) dioxane, pH 6.0 andequilibrated against 100× volume of 2.0 M ammonium sulfate, 0.1 M sodiumcitrate, 1% (v/v) dioxane, pH 6.0. After 24 hours of equilibration,crystal growth was induced by applying the seeding technique describedin ref. 21, using crystals of recombinant wild-type Bet v 1 (SEQ ID NO:37) as a source of seeds.

[0130] After about 2 months, crystals were harvested and analysed usingX-rays generated from a Rigaku rotating anode as described in ref. 21and the structure was solved using molecular replacement.

[0131] Structure of Bet v 1 Glu45Ser Mutant (SEQ ID NO: 37)

[0132] The structural effect of the mutation was addressed by growingthree-dimensional Bet v 1 Glu45Ser protein (SEQ ID NO: 37) crystalsdiffracting to 3.0 Å resolution when analysed by X-rays generated from arotating anode. The substitution of glutamic acid to serine in position45 was verified by the Bet v 1 Glu45Ser (SEQ ID NO: 37) structureelectron density map which also showed that the overall α-carbonbackbone tertiary structure is preserved.

[0133] IgE-Binding Properties of Bet v 1 Glu45Ser Mutant (SEQ ID NO: 37)

[0134] The IgE-binding properties of Bet v 1 Glu45Ser mutant (SEQ ID NO:37) was compared with recombinant Bet v 1 in a fluid-phaseIgE-inhibition assay using a pool of serum IgE derived from birchallergic patients.

[0135] Recombinant Bet v 1 no. 2801 (SEQ ID NO: 37) was biotinylated ata molar ratio of 1:5 (Bet v 1 no. 2801:biotin). The inhibition assay wasperformed as follows: a serum sample (25 μl) was incubated with solidphase anti IgE, washed, re-suspended and further incubated with amixture of biotinylated Bet v 1 no. 2801 (SEQ ID NO: 37) (3.4 nM) and agiven mutant (0-28.6 nM). The amount of biotinylated Bet v 1 no. 2801(SEQ ID NO: 37) bound to the solid phase was estimated from the measuredRLU after incubation with acridinium ester labelled streptavidin. Thedegree of inhibition was calculated as the ratio between the RLU'sobtained using buffer and mutant as inhibitor.

[0136]FIG. 4 shows the inhibition of the binding of biotinylatedrecombinant Bet v 1 (SEQ ID NO: 37) to serum IgE from a pool of allergicpatients by non-biotinylated Bet v 1 and by Bet v 1 Glu45Ser mutant (SEQID NO: 37).

[0137] There is a clear difference in the amount of respectiverecombinant proteins necessary to reach 50% inhibition of the binding toserum IgE present in the serum pool. Recombinant Bet v 1 (SEQ ID NO: 37)reaches 50% inhibition at about 6.5 ng whereas the correspondingconcentration for Bet v 1 Glu45Ser mutant (SEQ ID NO: 37) is about 12ng. This show that the point mutation introduced in Bet v 1 Glu45Sermutant (SEQ ID NO: 37) lowers the affinity for specific serum IgE by afactor of about 2.

[0138] The maximum level of inhibition reached by the Bet v 1 Glu45Sermutant (SEQ ID NO: 37) is clearly lower compared to recombinant Bet v 1.(SEQ ID NO: 37) This may indicate that after the Glu45Ser substitution(SEQ ID NO: 37), some of the specific IgE present in the serum pool areunable to recognise the Bet v 1 Glu45Ser mutant (SEQ ID NO: 37).

[0139] Bet v 1 Mutant Asn28Thr+Lys32Gln (SEQ ID NO: 37)

[0140] Aspartate and lysine in positions 28 and 32, respectively show ahigh degree of solvent-exposure (35% and 50%, respectively) and arelocated in a molecular surface patch common for Fagales allergens (patchII). In the structure, aspartate 28 and lysine 32 are located close toeach other on the molecular surface and most likely interact viahydrogen bonds. A threonine and a gluatamate residue were found tooccupy positions 28 and 32, respectively in some of the Bet v 1homologous PR-10 proteins arguing for that aspartate and lysine can bereplaced with threonine and glutamate, respectively without distortionof the α-carbon backbone tertiary structure. In addition, as none of thenaturally occurring isoallergen sequences have threonine and glutamatein positions 28 and 32, respectively, the substitutions gives rise to anon-naturally occurring Bet v 1 molecule (SEQ ID NO: 37).

[0141] IgE-Binding Properties of Bet v 1 Mutant Asn28Thr+Lys32Gln (SEQID NO: 37)

[0142] The IgE-binding properties of mutant Asn28Thr+Lys32Gln (SEQ IDNO: 37) was compared with recombinant Bet v 1 in a fluid-phaseIgE-inhibition assay using the pool of serum IgE derived from birchallergic patients described above.

[0143]FIG. 5 shows the inhibition of the binding of biotinylatedrecombinant Bet v 1 (SEQ ID NO: 37) to serum IgE from a pool of Allergicpatients by non-biotinylated Bet v 1 and by Bet v 1 mutantAsn28Thr+Lys32Gln (SEQ ID NO: 37).

[0144] There is a clear difference in the amount of respectiverecombinant proteins necessary to reach 50% inhibition of the binding toserum IgE present in the serum pool. Recombinant Bet v 1 reaches 50%inhibition at about 6.5 ng whereas the corresponding concentration forBet v 1 mutant Asn28Thr+Lys32Gln (SEQ ID NO: 37) is about 12 ng. Thisshow that the point mutations introduced in Bet v 1 (SEQ ID NO: 37)mutant Asn28Thr+Lys32Gln (SEQ ID NO: 37) lowers the affinity forspecific serum IgE by a factor of about 2.

[0145] The maximum level of inhibition reached by the Bet v 1 mutantAsn28Thr+Lys32Gln mutant (SEQ ID NO: 37) is clearly lower compared torecombinant Bet v 1. This may indicate that after the Asn28Thr+Lys32Glnsubstitutions, some of the specific IgE present in the serum pool areunable to recognise the Bet v 1 mutant Asn28Thr+Lys32Gln (SEQ ID NO:37).

[0146] Bet v 1 Mutant Pro108Gly (SEQ ID NO: 37)

[0147] Proline in position 108 show a high degree of solvent-exposure(60%) and is located in a molecular surface patch common for Fagalesallergens (patch III). A glycine residue was found to occupy position108 in some of the Bet v 1 homologous PR-10 proteins arguing for thatproline can be replaced with glycine without distortion of the α-carbonbackbone tertiary structure. In addition, as none of the naturallyoccurring isoallergen sequences have glycine in position 108, thesubstitution of proline with glycine gives rise to a non-naturallyoccurring Bet v 1 molecule (SEQ ID NO: 37).

[0148] IgE-Binding Properties of Bet v 1 Pro108Gly Mutant (SEQ ID NO:37)

[0149] The IgE-binding properties of Bet v 1 Pro108Gly mutant (SEQ IDNO: 37) was compared with recombinant Bet v 1 (SEQ ID NO: 37) in afluid-phase IgE-inhibition assay using the pool of serum IgE derivedfrom birch allergic patients described above.

[0150]FIG. 6 shows the inhibition of the binding of biotinylatedrecombinant Bet v 1 (SEQ ID NO: 37) to serum IgE from a pool of allergicpatients by non-biotinylated Bet v 1 (SEQ ID NO: 37) and by Bet v 1Pro108Gly mutant (SEQ ID NO: 37).

[0151] There is a clear difference in the amount of respectiverecombinant proteins necessary to reach 50% inhibition of the binding toserum IgE present in the serum pool. Recombinant Bet v 1 reaches 50%inhibition at about 6.5 ng whereas the corresponding concentration forBet v 1 Pro108Gly (SEQ ID NO: 37) is 15 ng. This show that the singlepoint mutation introduced in Bet v 1 Pro108Gly (SEQ ID NO: 37) lowersthe affinity for specific serum IgE by a factor of about 2.

[0152] The maximum level of inhibition reached by the Bet v 1 Pro108Glymutant (SEQ ID NO: 37) is somewhat lower compared to recombinant Bet v 1(SEQ ID NO: 37). This may indicate that after the Pro108Glysubstitution, some of the specific IgE present in the serum pool areunable to recognise the Bet v 1 Pro108Gly mutant (SEQ ID NO: 37).

[0153] Bet v 1 Mutant Glu60Ser (Non-Patch Mutant) (SEQ ID NO: 37)

[0154] Glutamic acid in position 60 show a high degree ofsolvent-exposure (60%) however, it is not located in a molecular surfacepatch common for Fagales allergens. A serine residue was found to occupyposition 60 in some of the Bet v 1 homologous PR-10 proteins arguing forthat glutamic acid can be replaced with serine without distortion of theα-carbon backbone tertiary structure. In addition, as none of thenaturally occurring isoallergen sequences have serine in position 60,the substitution of glutamic acid with serine gives rise to anon-naturally occurring Bet v 1 molecule (SEQ ID NO: 37).

[0155] IgE-Binding Properties of Bet v 1 Glu60Ser Mutant (SEQ ID NO: 37)

[0156] The IgE-binding properties of Bet v 1 Glu60Ser mutant (SEQ ID NO:37) was compared with recombinant Bet v 1 in a fluid-phaseIgE-inhibition assay using the pool of serum IgE derived from birchallergic patients described above.

[0157]FIG. 7 shows the inhibition of the binding of biotinylatedrecombinant Bet v 1 (SEQ ID NO: 37) to serum IgE from a pool of allergicpatients by non-biotinylated Bet v1 (SEQ ID NO: 37) and by Bet v1Glu60Ser mutant (SEQ ID NO: 37). In contrast to the Glu45Ser, Pro108Glyand Asn28Thr+Lys32Gln mutants (SEQ ID NO: 37), the substitution glutamicacid 60 to serine, does not show any significant effect on theIgE-binding properties of Bet v 1 Glu60Ser mutant (SEQ ID NO: 37). Thisindicates that substitutions outside the defined Fagales common patchesonly have a marginal effect on the binding of specific serum IgEsupporting the concept that conserved allergen molecular surface areasharbor dominant IgE-binding epitopes.

[0158] Bet v 1 Triple-Patch Mutant (SEQ ID NO: 37)

[0159] In the Triple-patch mutant, the point mutations (Glu45Ser,Asn28Thr+Lys32Gln and Pro108Gly) introduced in the three differentcommon Fagales patches, described above, were simultaneously introducedin creating an artificial mutant carrying four amino acid substitutions.

[0160] Structural Analysis of Bet v 1 Triple-Patch Mutant (SEQ ID NO:37)

[0161] The structural integrity of the purified Triple-patch mutant wasanalysed by circular dichroism (CD) spectroscopy. FIG. 8 shows the CDspectra of recombinant and Triple-patch mutant (SEQ ID NO: 37), recordedat close to equal concentrations. The overlap in peak amplitudes andpositions in the CD spectra from the two recombinant proteins shows thatthe two preparations contain equal amounts of secondary structuresstrongly suggesting that the α-carbon backbone tertiary structure is notaffected by the introduced amino acid substitutions.

[0162] IgE-Binding Properties of Bet v 1 Triple-Patch Mutant (SEQ ID NO:37)

[0163] The IgE-binding properties of Bet v 1 Triple-patch mutant (SEQ IDNO: 37) was compared with recombinant Bet v 1 (SEQ ID NO: 37) in afluid-phase IgE-inhibition assay using the pool of serum IgE derivedfrom birch allergic patients described above.

[0164]FIG. 9 shows the inhibition of the binding of biotinylatedrecombinant Bet v 1 (SEQ ID NO: 37) to serum IgE from a pool of allergicpatients by non-biotinylated Bet v 1 (SEQ ID NO: 37) and by Bet v 1Triple-patch mutant (SEQ ID NO: 37). In contrast to the single mutantsdescribed above, the inhibition curve of the Triple-patch mutant (SEQ IDNO: 37) is no longer parallel relative to recombinant. This shows thatthe substitutions introduced in the Triple-patch mutant (SEQ ID NO: 37)has changed the IgE-binding properties and epitope profile compared torecombinant. The lack of parallellity makes it difficult to quantify thedecrease of the Triple-patch mutant (SEQ ID NO: 37) affinity forspecific serum IgE.

[0165] Recombinant Bet v 1 (SEQ ID NO: 37) reaches 50% inhibition atabout 6 ng whereas the corresponding concentration for Bet v 1Triple-patch mutant (SEQ ID NO: 37) is 30 ng, i.e a decrease in affinityby a factor 5. However, in order to reach 80% inhibition thecorresponding values are 20 ng and 400 ng, respectively, i.e a decreaseby a factor 20.

[0166] T Cell Proliferation Assay Using Recombinant Bet v 1 Triple-PatchMutant (SEQ ID NO: 37)

[0167] The analysis was carried out as described in ref. 15. It wasfound that recombinant Bet v 1 Triple-patch mutant (SEQ ID NO: 37) wasable to induce proliferation in T cell lines from three different birchpollen allergic patients with stimulation indices similar to recombinantand naturally occurring. This suggests that the Triple-patch mutant (SEQID NO: 37) can initiate the cellular immune response necessary forantibody production.

Example 2

[0168] Identification of Common Epitopes within Vespula vulgaris VenomMajor Allergen Antigen 5 (SEQ ID NOS: 38-39)

[0169] Antigen 5 is one of the three vespid venom proteins, which areknown allergens in man. The vespids include hornets, yellow-jacket andwasps. The other two known allergens of vespid venoms are phospholipaseA₁ and hyaluronidase. Antigen 5 from Vespula vulgaris (Ves v 5) (SEQ IDNO: 39) has been cloned and expressed as recombinant protein in theyeast system (Monsalve et al. 1999, ref. 22). The three-dimensionalcrystal structure of recombinant Ves v 5 (SEQ ID NO: 39) has recentlybeen determined at 1.8 Å resolution (in preparation). The main featuresof the structure consist of four β-strands and four α-helices arrangedin three stacked layers giving rise to a “α-β-α sandwich”. The sequenceidentity between Antigen 5 homologous allergens from different Vespulaspecies is about 90% suggesting presence of conserved molecular surfaceareas and B cell epitopes.

[0170] The presence and identification of common patches was performedafter alignment of all known amino acid sequences, as previouslydescribed for tree pollen allergens, of the Vespula antigen 5 allergensin combination with an analysis of the molecular surface of Antigen 5revealed by the three-dimensional structure of Ves v 5 (SEQ ID NO: 39).FIG. 10 shows solvent accessibility of individually aligned antigen 5residues and alignment of Vespula antigen 5 sequences (left panel). Onthe right panel of FIG. 10 is shown the molecular surface of antigen 5(SEQ ID NO: 39) with conserved areas among Vespula antigen 5:s coloured.

[0171] Selection of Amino Acid Residues for Site-Directed Mutagenesis

[0172] Amino acid residues for site-directed mutagenesis were selectedamong residues present the patches common for Vespula sincemodifications of these is expected to affect the binding of serum IgEfrom the majority of patients showing clinical Vespula allergiccross-reactivity.

[0173] The relative orientation and percentage of solvent-exposure ofeach amino acid residue within respective patch was calculated based ontheir atomic coordinates. Residues having a low degree of solventexposure were not regarded suitable for mutagenesis due to the possibledisruption of the structure or lack of antibody interaction. Theremaining residues were ranked according to their degree ofsolvent-exposure.

[0174] Cloning of the Gene Encoding Ves v 5 (SEQ ID NO: 38)

[0175] Total RNA was isolated from venom acid glands of Vespula vulgarisvespids as described in (Fang et al. 1988, ref. 23).

[0176] First-strand cDNA synthesis, PCR amplification and cloning of theVes v 5 (SEQ ID NO: 38) gene was performed as described in (Lu et al.1993, ref. 24)

[0177] Subcloning into pPICZαA

[0178] The gene encoding Ves v 5 (SEQ ID NO: 38) was subsequentlysub-cloned into the pPICZαA vector (Invitrogen) for secreted expressionof Ves v 5 in Pichia pastoris (SEQ ID NO: 39). The gene was amplified byPCR and sub-cloned in frame with the coding sequence for the α-factorsecretion signal of Saccharomyces cerevisiae. In this construct theα-factor is cleaved off, in vivo, by the Pichia pastoris Kex2 proteasesystem during secretion of the protein.

[0179] In brief PCR was performed using Ves v 5 (SEQ ID NO: 38) astemplate and primers corresponding to the amino- and carboxyterminus ofthe protein (SEQ ID NO: 39), respectively. The primers were extended inthe 5′-end to accommodate restriction sites for cloning, EcoRI and XbaI,respectively. Nucleotides encoding the Kex2 cleavage site was in thisconstruct positioned 18 nucleotides upstream to the amino terminus ofthe protein (SEQ ID NO: 39), resulting in the expression of Ves v 5 withsix additional amino acids, Glu-Ala-Glu-Ala-Glu-Phe, at the aminoterminus (SEQ ID NO: 39).

[0180] Insertion of pPICZαA-Ves v 5 into P. pastoris

[0181] The pPICZαA vectors with the Ves v 5 gene (SEQ ID NO: 38)inserted was linearised by Sac I restriction and inserted into the AOX1locus on the Pichia pastoris genome. Insertion was performed byhomologous recombination on Pichia pastoris KM71 cells following therecommendations of Invitrogen.

[0182] In Vitro Mutagenesis

[0183] In vitro mutagenesis was performed by PCR using recombinantpPICZαA with Ves v 5 inserted as template. Each mutant Ves v 5 gene (SEQID NO: 38) was generated by 3 PCR reactions using 4 primers.

[0184] Two mutation-specific oligonucleotide primers were synthesisedaccommodating each mutation, one for each DNA strand, see FIGS. 11 and12 (SEQ ID NOS: 23-35 and 40). Using the mutated nucleotide(s) asstarting point both primers were extended 6-7 nucleotides in the 5′-endand 12-13 nucleotides in the 3′-end. The extending nucleotides wereidentical in sequence to the Ves v 5 gene (SEQ ID NO: 38) in the actualregion.

[0185] Two generally applicable primers (denoted “all sense” (SEQ ID NO:40) and “all non-sense” (SEQ ID NO: 35) in FIG. 12) were furthermoresynthesised and used for all mutants. To insure expression of Ves v 5mutants with authentic amino terminus, one primer corresponding to theamino terminus of the protein was extended in the 5′-end with a Xho Isite. Upon insertion of the Ves v 5 mutant genes (SEQ ID NO: 38) intothe pPICZαA vector, the Kex2 protease cleavage site was regenerateddirectly upstream to the amino terminus of Ves v 5 (SEQ ID NO: 39). Thesecond primer was corresponding in sequence to a region of the pPICZαAvector positioned approximately 300 bp downstream from the Ves v 5 gene(SEQ ID NO: 38). The sequence of the primer corresponding to the aminoterminus of Ves v 5 (SEQ ID NO: 39) is derived from the sense strand andthe sequence of the downstream primer is derived from the non-sensestrand, see FIG. 11 (SEQ ID NOS: 23-30).

[0186] Two independent PCR reactions were performed essentiallyaccording to standard procedures (Saiki et al 1988) with the exceptionthat only 20 temperature cycles were performed in order to reduce thefrequency of PCR artefacts. Each PCR reaction used pPICZαA with Ves v 5(SEQ ID NO: 38) inserted as template and one mutation-specific and onegenerally applicable primer in meaningful combinations.

[0187] The PCR products were purified by using “Concert, Rapid PCRPurification System” (Life Technologies). A third PCR reaction wasperformed using the combined PCR products from the first two PCRreactions as template and both generally applicable primers. Again, 20cycles of standard PCR were used. The PCR product was purified with the“Concert, Rapid PCR Purification System” (Life Technologies), cut withrestriction enzymes (XhoI/XbaI), and ligated directionally into pPICZαAvector restricted with the same enzymes. FIG. 13 shows an overview ofall Ves v 5 mutations (SEQ ID NOS: 38-39).

[0188] Insertion of pPICZαA-Ves v 5 Mutants into P. pastoris

[0189] The pPICZαA vectors with the Ves v 5 mutant genes (SEQ ID NO: 38)inserted were linearised by Sac I restriction and inserted into the AOX1locus on the Pichia pastoris genome. Insertions were performed byhomologous recombination on Pichia pastoris KM71 cells following therecommendations of Invitrogen.

[0190] Nucleotide Sequencing

[0191] Determination of the nucleotide sequence of the Ves v 5 encodinggene (SEQ ID NO: 38) was performed before and after subcloning, andfollowing in vitro mutagenesis, respectively.

[0192] Plasmid DNA's from 10 ml of bacterial culture grown to saturationovernight in LB medium supplemented with 0.1 g/l ampicillin werepurified on Qiagen-tip 20 columns and sequenced using the Sequenaseversion 2.0 DNA sequencing kit (USB) following the recommendations ofthe suppliers.

[0193] Expression and Purification of Recombinant Ves v 5 (SEQ ID NO:39)

[0194] Recombinant yeast cells of Pichia pastoris strain KM71 were grownin 500 ml bottles containing 100 ml of pH 6.0 phosphate buffercontaining yeast nitrogen base, biotin, glycerol and histidine at 30° C.with orbital shaking at 225 rpm until A₆₀₀ nm of 4-6. Cells werecollected by centrifugation and re-suspended in 10 ml of similarbuffered medium containing methanol in place of glycerol. Incubation wascontinued at 30° C. for 7 days with daily addition of 0.05 ml methanol.

[0195] Cells were harvested by centrifugation and the collected culturefluid was concentrated by ultrafiltration. After dialysis against 50 mMammonium acetate buffer, pH 4.6, the sample was applied to a FPLC(Pharmacia) SE-53 cation exchange column equilibrated in the samebuffer. The column was eluated with a 0-1.0 M NaCl, 50 mM ammoniumacetate linear gradient. The recombinant Ves v 5 peak eluting at about0.4 M NaCl was collected and dialysed against 0.02 N acetic acid. Afterconcentration to about 10 mg/ml, the purified Ves v 5 (SEQ ID NO: 39)was stored at 4° C.

[0196] Crystallisation of Recombinant Ves v 5 (SEQ ID NO: 39)

[0197] Crystals of Ves v 5 (SEQ ID NO: 39) was grown by the vapourdiffusion technique at 25° C. For crystallisation, 5 μl of 5 mg/ml Ves v5 was mixed with 5 μl of 18% PEG 6000, 0.1 M sodium citrate, pH 6.0 andequilibrated against 1 ml of 18% PEG 6000, 0.1 M sodium citrate, pH 6.0.

[0198] X-ray diffraction data was collected at 100K from native Ves v 5crystals (SEQ ID NO: 39) and after incorporation of heavy-atomderivatives and used to solve the three-dimensional structure of Ves v 5(SEQ ID NO: 39), see FIG. 10 (manuscript in preparation).

[0199] Immunoelectrophoresis Using Rabbit Polyclonal Antibodies

[0200] The two Ves v 5 mutants were produced as recombinant Ves v 5proteins (SEQ ID NO: 39) and tested for their reactivity towardspolyclonal rabbit antibodies raised against recombinant Ves v 5. Whenanalysed by rocket immunoelectrophoresis under native conditions, therabbit antibodies were able to precipitate recombinant Ves v 5 (SEQ IDNO: 39) as well as both mutants, indicating that the mutants haveconserved α-carbon backbone tertiary structure.

[0201] Inhibition of Specific serum IgE

[0202] The IgE-binding properties of Ves v 5 mutants (SEQ ID NO: 39)were compared to recombinant Ves v 5 (SEQ ID NO: 39) in a fluid-phaseIgE-inhibition assay using a pool of serum IgE derived from vespid venomallergic patients.

[0203] The inhibition assay was performed as described above usingbiotinylated recombinant Ves v 5 (SEQ ID NO: 39) instead of Bet v 1 (SEQID NO: 37).

[0204] Ves v 5 Lys72Ala Mutant

[0205] Lysine in position 72 show a high degree of solvent-exposure(70%) and is located in a molecular surface patch common for Vespulaantigen 5. The relative orientation and high degree of solvent exposureargued for that lysine 72 can be replaced by an alanine residue withoutdistortion of the α-carbon backbone tertiary structure. In addition, asnone of the naturally occurring isoallergen sequences have alanine inposition 72, the substitution of lysine with alanine gives rise to anon-naturally occurring Ves v 5 molecule.

[0206] IgE-Binding Properties of Ves v 5 Lys72Ala Mutant (SEQ ID NO: 39)

[0207] The IgE-binding properties of Ves v 5 Lys72Ala mutant (SEQ ID NO:39) was compared with recombinant Ves v 5 in a fluid-phaseIgE-inhibition assay using the pool of serum IgE derived from birchallergic patients described above.

[0208]FIG. 14 shows the inhibition of the binding of biotinylatedrecombinant Ves v 5 (SEQ ID NO: 39) to serum IgE from a pool of allergicpatients by non-biotinylated Ves v 5 (SEQ ID NO: 39) and by Ves v 5Lys72Ala mutant (SEQ ID NO: 39).

[0209] There is a clear difference in the amount of respectiverecombinant proteins necessary to reach 50% inhibition of the binding toserum IgE present in the serum pool. Recombinant Ves v 5 (SEQ ID NO: 39)reaches 50% inhibition at about 6 ng whereas the correspondingconcentration for Ves v 5 Lys72Ala mutant (SEQ ID NO: 39) is 40 ng. Thisshow that the single point mutation introduced in Ves v 5 Lys72Alamutant (SEQ ID NO: 39) lowers the affinity for specific serum IgE by afactor of about 6.

[0210] The maximum level of inhibition reached by the Ves v 5 Lys72Alamutant (SEQ ID NO: 39) significantly lower compared to recombinant Ves v5 (SEQ ID NO: 39). This may indicate that after the Lys72Alasubstitution, some of the specific IgE present in the serum pool areunable to recognise the Ves v 5 Lys72Ala mutant (SEQ ID NO: 39).

[0211] Ves v 5 Tyr96Ala Mutant (SEQ ID NO: 39)

[0212] Tyrosine in position 96 show a high degree of solvent-exposure(65%) and is located in a molecular surface patch common for Vespulaantigen 5 (SEQ ID NO: 39). The relative orientation an high degree ofsolvent exposure argued for that tyrosine 96 can be replaced by analanine residue without distortion of the three-dimensional structure.In addition, as none of the naturally occurring isoallergen sequenceshave alanine in position 96, the substitution of tyrosine with alaninegives rise to a non-naturally occurring Ves v 5 molecule (SEQ ID NO:39).

[0213] IgE-Binding Properties of Ves v 5 Tyr96Ala Mutant (SEQ ID NO: 39)

[0214] The IgE-binding properties of Ves v 5 Tyr96Ala mutant (SEQ ID NO:39) was compared with recombinant Ves v 5 in a fluid-phaseIgE-inhibition assay using the pool of serum IgE derived from birchallergic patients described above.

[0215]FIG. 14 shows the inhibition of the binding of biotinylatedrecombinant Ves v 5 (SEQ ID NO: 39) to serum IgE from a pool of allergicpatients by non-biotinylated Ves v 5 (SEQ ID NO: 39) and by Ves v 5Tyr96Ala mutant (SEQ ID NO: 39).

[0216] There is a clear difference in the amount of respectiverecombinant proteins necessary to reach 50% inhibition of the binding toserum IgE present in the serum pool. Recombinant Ves v 5 (SEQ ID NO: 39)reaches 50% inhibition at about 6 ng whereas the correspondingconcentration for Ves v 5 Tyr96Ala mutant (SEQ ID NO: 39) is 40 ng.

[0217] This show that the single point mutation introduced in Ves v 5Tyr96Ala mutant (SEQ ID NO: 39) lowers the affinity for specific serumIgE by a factor of about 6.

[0218] The maximum level of inhibition reached by the Ves v 5 Tyr96Alamutant (SEQ ID NO: 39) significantly lower compared to recombinant Ves v5 (SEQ ID NO: 39). This may indicate that after the Tyr96Alasubstitution, some of the specific IgE present in the serum pool areunable to recognise the Ves v 5 Tyr96Ala mutant (SEQ ID NO: 39).

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1 40 1 41 DNA Artificial Sequence primer 1 aattatgaga ctgagaccacctctgttatc ccagcagctc g 41 2 41 DNA Artificial Sequence primer 2ttaatactct gactctggtg gagacaatag ggtcgtcgag c 41 3 23 DNA ArtificialSequence primer 3 tgagaccccc tctgttatcc cag 23 4 23 DNA ArtificialSequence primer 4 atactctgac tctgggggag aca 23 5 15 DNA ArtificialSequence primer 5 gttgccaacg atcag 15 6 23 DNA Artificial Sequenceprimer 6 tgagaccccc tctgttatcc cag 23 7 23 DNA Artificial Sequenceprimer 7 acagaggggg tctcagtctc ata 23 8 31 DNA Artificial Sequenceprimer 8 gataccctct ttccacaggt tgcaccccaa g 31 9 31 DNA ArtificialSequence primer 9 acctgtggaa agagggtatc gccatcaagg a 31 10 23 DNAArtificial Sequence primer 10 aacatttcag gaaatggagg gcc 23 11 23 DNAArtificial Sequence primer 11 tttcctgaaa tgttttcaac act 23 12 23 DNAArtificial Sequence primer 12 ttaagaacat cagctttccc gaa 23 13 23 DNAArtificial Sequence primer 13 agctgatgtt cttaatggtt cca 23 14 23 DNAArtificial Sequence primer 14 ggaccatgca aacttcaaat aca 23 15 23 DNAArtificial Sequence primer 15 agtttgcatg gtccacctca tca 23 16 23 DNAArtificial Sequence primer 16 tttccctcag gcctcccttt caa 23 17 23 DNAArtificial Sequence primer 17 aggcctgagg gaaagctgat ctt 23 18 24 DNAArtificial Sequence primer 18 tgaaggatct ggagggcctg gaac 24 19 24 DNAArtificial Sequence primer 19 ccctccagat ccttcaatgt tttc 24 20 24 DNAArtificial Sequence primer 20 ggcaactggt gatggaggat ccat 24 21 24 DNAArtificial Sequence primer 21 ccatcaccag ttgccactat cttt 24 22 15 DNAArtificial Sequence primer 22 catgccatcc gtaag 15 23 41 DNA ArtificialSequence primer 23 accacagcct ccagcgaaga atatgaaaaa tttggtatgg a 41 2441 DNA Artificial Sequence primer 24 tggtgtcgga ggtcgcttct tatactttttaaaccatacc t 41 25 21 DNA Artificial Sequence primer 25 ccagcggctaatatgaaaaa t 21 26 21 DNA Artificial Sequence primer 26 gtcggaggtcgccgattata c 21 27 41 DNA Artificial Sequence primer 27 ggctaatcaatgtcaatatg gtcacgatac ttgcagggat g 41 28 41 DNA Artificial Sequenceprimer 28 ccgattagtt acagttatac cagtgctatg aacgtcccta c 41 29 21 DNAArtificial Sequence primer 29 tgtcaagctg gtcacgatac t 21 30 21 DNAArtificial Sequence primer 30 ttagttacag ttcgaccagt g 21 31 21 DNAArtificial Sequence primer 31 ccagcggcta atatgaaaaa t 21 32 21 DNAArtificial Sequence primer 32 catattagcc gctggaggct g 21 33 21 DNAArtificial Sequence primer 33 tgtcaagctg gtcacgatac t 21 34 21 DNAArtificial Sequence primer 34 gtgaccagct tgacattgat t 21 35 21 DNAArtificial Sequence primer 35 attcatcagc tgcgagatag g 21 36 480 DNAbetula verrucosa 36 ggtgtgttta attatgagac tgagaccacc tctgttatcccagcagctcg actgttcaag 60 gcctttatcc ttgatggcga taacctcttt ccaaaggttgcaccccaagc cattagcagt 120 gttgaaaaca ttgaaggaaa tggagggcct ggaaccattaagaagatcag ctttcccgaa 180 ggcctccctt tcaagtacgt gaaggacaga gttgatgaggtggaccacac aaacttcaaa 240 tacaattaca gcgtgatcga gggcggtccc ataggcgacacattggagaa gatctccaac 300 gagataaaga tagtggcaac ccctgatgga ggatccatcttgaagatcag caacaagtac 360 cacaccaaag gtgaccatga ggtgaaggca gagcaggttaaggcaagtaa agaaatgggc 420 gagacacttt tgagggccgt tgagagctac ctcttggcacactccgatgc ctacaactaa 480 37 159 PRT betula verrucosa 37 Gly Val Phe AsnTyr Glu Thr Glu Thr Thr Ser Val Ile Pro Ala Ala 1 5 10 15 Arg Leu PheLys Ala Phe Ile Leu Asp Gly Asp Asn Leu Phe Pro Lys 20 25 30 Val Ala ProGln Ala Ile Ser Ser Val Glu Asn Ile Glu Gly Asn Gly 35 40 45 Gly Pro GlyThr Ile Lys Lys Ile Ser Phe Pro Glu Gly Leu Pro Phe 50 55 60 Lys Tyr ValLys Asp Arg Val Asp Glu Val Asp His Thr Asn Phe Lys 65 70 75 80 Tyr AsnTyr Ser Val Ile Glu Gly Gly Pro Ile Gly Asp Thr Leu Glu 85 90 95 Lys IleSer Asn Glu Ile Lys Ile Val Ala Thr Pro Asp Gly Gly Ser 100 105 110 IleLeu Lys Ile Ser Asn Lys Tyr His Thr Lys Gly Asp His Glu Val 115 120 125Lys Ala Glu Gln Val Lys Ala Ser Lys Glu Met Gly Glu Thr Leu Leu 130 135140 Arg Ala Val Glu Ser Tyr Leu Leu Ala His Ser Asp Ala Tyr Asn 145 150155 38 615 DNA vespula vulgaris 38 aacaattatt gtaaaataaa atgtttgaaaggaggtgtcc atactgcctg caaatatgga 60 agtcttaaac cgaattgcgg taataaggtagtggtatcct atggtctaac gaaacaagag 120 aaacaagaca tcttaaagga gcacaatgactttagacaaa aaattgcacg aggattggag 180 actagaggta atcctggacc acagcctccagcgaagaata tgaaaaattt ggtatggaac 240 gacgagttag cttatgtcgc ccaagtgtgggctaatcaat gtcaatatgg tcacgatact 300 tgcagggatg tagcaaaata tcaggttggacaaaacgtag ccttaacagg tagcacggct 360 gctaaatacg atgatccagt taaactagttaaaatgtggg aagatgaagt gaaagattat 420 aatcctaaga aaaagttttc gggaaacgactttctgaaaa ccggccatta cactcaaatg 480 gtttgggcta acaccaagga agttggttgtggaagtataa aatacattca agagaaatgg 540 cacaaacatt accttgtatg taattatggacccagcggaa actttaagaa tgaggaactt 600 tatcaaacaa agtaa 615 39 204 PRTvespula vulgaris 39 Asn Asn Tyr Cys Lys Ile Lys Cys Leu Lys Gly Gly ValHis Thr Ala 1 5 10 15 Cys Lys Tyr Gly Ser Leu Lys Pro Asn Cys Gly AsnLys Val Val Val 20 25 30 Ser Tyr Gly Leu Thr Lys Gln Glu Lys Gln Asp IleLeu Lys Glu His 35 40 45 Asn Asp Phe Arg Gln Lys Ile Ala Arg Gly Leu GluThr Arg Gly Asn 50 55 60 Pro Gly Pro Gln Pro Pro Ala Lys Asn Met Lys AsnLeu Val Trp Asn 65 70 75 80 Asp Glu Leu Ala Tyr Val Ala Gln Val Trp AlaAsn Gln Cys Gln Tyr 85 90 95 Gly His Asp Thr Cys Arg Asp Val Ala Lys TyrGln Val Gly Gln Asn 100 105 110 Val Ala Leu Thr Gly Ser Thr Ala Ala LysTyr Asp Asp Pro Val Lys 115 120 125 Leu Val Lys Met Trp Glu Asp Glu ValLys Asp Tyr Asn Pro Lys Lys 130 135 140 Lys Phe Ser Gly Asn Asp Phe LeuLys Thr Gly His Tyr Thr Gln Met 145 150 155 160 Val Trp Ala Asn Thr LysGlu Val Gly Cys Gly Ser Ile Lys Tyr Ile 165 170 175 Gln Glu Lys Trp HisLys His Tyr Leu Val Cys Asn Tyr Gly Pro Ser 180 185 190 Gly Asn Phe LysAsn Glu Glu Leu Tyr Gln Thr Lys 195 200 40 38 DNA Artificial Sequenceprimer 40 ccgctcgaga aaagaaacaa ttattgtaaa ataaaatg 38

1. A method of preparing a recombinant allergen derived from a naturallyoccurring allergen, wherein specific IgE binding to the recombinantallergen is reduced compared to the IgE binding to said naturallyoccurring allergen, which method comprises: a) identifying amino acidresidues in said naturally occurring allergen which are conserved withmore than 70% identity in all of the known homologous proteins withinthe taxonomic order from which said naturally occurring allergenoriginates; b) defining at least one patch of said conserved amino acidresidues which patch (i) is connected over at least 400 Å² of thesurface of the three-dimensional structure of said naturally occurringallergen molecule; and (ii) comprises at least one B cell epitope, andwherein (iii) said conserved amino acid residues have a solventaccessibility of at least 20%; and c) substituting at least one of saididentified amino acid residues in said defined at least one patch with anon-conservative amino acid residue, wherein the α-carbon backbonetertiary structure of the recombinant allergen is essentially preservedas compared with the α-carbon backbone tertiary structure of saidnaturally occurring allergen.
 2. The method of claim 1, wherein theamino acid residues identified in step (a) are 90% or more conserved. 3.The method of claim 1, which method further comprises: b′) ranking saidconserved amino acid residues within said at least one patch in order ofpercent solvent accessibility; and b″) selecting said at least one aminoacid residue for substitution in step (c) from the amino acid residueshaving a solvent accessibility of 20-80%.
 4. The method of claim 1,wherein the percent solvent accessibility is in a range from 30-80%. 5.The method of claim 1 in which the average root mean square of theatomic coordinates of the α-carbon backbone tertiary structure of therecombinant allergen deviates from the average root mean square of theatomic coordinates of the α-carbon backbone tertiary structure of saidnaturally occurring allergen by less than 2 Å.
 6. The method of claim 1,wherein the amino acid residue substitution in step (c) is performedusing site-directed mutagenesis.
 7. The method of claim 1, wherein saidat least one amino acid residue being substituted is a polar amino acidand said non-conservative amino acid is a polar amino acid.
 8. Themethod of claim 1, wherein said at least one amino acid residue beingsubstituted is a non-polar amino acid and said non-conservative aminoacid is a non-polar amino acid.
 9. The method of claim 1, wherein saidnaturally occurring allergen is an inhalation allergen or venomallergen.
 10. The method of claim 1, wherein said naturally occurringallergen is a tree pollen allergen, grass pollen allergen, weed pollenallergen, herb pollen allergen, fungal allergen, dust mite allergen,animal allergen or insect allergen.
 11. The method of claim 10 wherein(a) said tree pollen allergen is from a tree taxonomically belonging toFagales, Oleales, or Pineales; (b) said grass pollen allergen is from agrass taxonomically belonging to Poales; (c) said herb pollen allergenis from an herb taxonomically belonging to Aasterales; (d) said fungalallergen is from a fungus taxonomically belonging to Alternaria orCladosporium; (e) said dust mite allergen is from a dust mitetaxonomically belonging to Dermatophagoides; (f) said animal allergen isfrom an animal taxonomically belonging to Felis, Canis, or Equus; (g)said insect allergen is from an insect taxonomically belonging toBlatella, Periplaneta, or Hymenoptera; (h) said weed pollen allergen isfrom a weed taxonomically belonging to Urticales.
 12. The method ofclaim 11 wherein (a) said tree taxonomically belonging to Fagalesbelongs taxonomically to Betula, Alnus, Corylus, or Carpinus; (b) saidtree taxonomically belonging to Oleales belongs taxonomically to Olea;(c) said tree taxonomically belonging to Pineales belongs taxonomicallyto Cryptomeria; (d) said grass taxonomically belonging to Poales belongstaxonomically to Cynodon, Dactylis, Festuca, Holcus, Lolium, Phleum,Poa, Secale or Sorghum; (e) said herb taxonomically belonging toAasterales belongs taxonomically to Ambrosia, Artemicia, or Urticales;(f) said fungus taxonomically belonging to Alternaria belongstaxonomically to Alternata; (g) said fungus taxonomically belonging toCladosporium belongs taxonomically to herbarium; (h) said dust mitebelonging taxonomically belonging to Dermatophagoides belongstaxonomically to Farinae or Pteronyssinus; (i) said animal taxonomicallybelonging to Felis belongs taxonomically to Domesticus; (j) said animaltaxonomically belonging to Canis belongs taxonomically to familiaris;(k) said animal taxonomically belonging to Equus belongs taxonomicallyto caballus; (l) said insect taxonomically belonging to Blatella belongstaxonomically to Germanica; (m) said insect taxonomically belonging toPeriplaneta belongs taxonomically to americana; (n) said insecttaxonomically belonging to Hymenoptera belongs taxonomically toVespidae, Apidae; and (o) said weed taxonomically belonging to Urticalesbelongs taxonomically to Parietaria.
 13. A recombinant allergen derivedfrom a naturally occurring allergen, wherein at least onesolvent-accessible amino acid which is essentially conserved with morethan 70% identity in all of the known homologous proteins within thetaxonomic order from which said naturally occurring allergen originates,is substituted with an amino acid residue that is not conserved inhomologous naturally-occurring allergens within said taxonomic order,wherein the α-carbon backbone tertiary structure of the recombinantallergen is conserved as compared with said naturally occurringallergen, and wherein specific IgE binding to the mutant allergen isreduced compared to IgE binding to said naturally-occurring allergen.14. The recombinant allergen of claim 13, wherein said at least onesolvent-accessible amino acid residue has a solvent accessibility of20-80%.
 15. The recombinant allergen of claim 13, in which between 1 and5 solvent-accessible amino acid residues per at least 400 Å², aresubstituted.
 16. The recombinant allergen of claim 13, wherein theaverage root mean square deviation of the atomic coordinates comparingthe α-carbon backbone tertiary structures of said recombinant and saidnaturally occurring allergen is less than 2 Å.
 17. The recombinantallergen of claim 13, wherein the specific IgE binding to saidrecombinant allergen is reduced by at least 5-10%.
 18. The recombinantallergen of claim 17, wherein the specific IgE binding to saidrecombinant allergen is reduced by at least 10%.
 19. The recombinantallergen of claim 13, wherein said naturally occurring allergen is aninhalation allergen or venom allergen.
 20. The recombinant allergen ofclaim 13, wherein said naturally occurring allergen is a tree pollenallergen, grass pollen allergen, weed pollen allergen, herb pollenallergen, fungal allergen, dust mite allergen, animal allergen or insectallergen.
 21. The recombinant allergen of claim 20 wherein (a) said treepollen allergen is from a tree taxonomically belonging to Fagales,Oleales, or Pineales; (b) said grass pollen allergen is from a grasstaxonomically belonging to Poales; (c) said herb pollen allergen is froman herb taxonomically belonging to Aasterales; (d) said fungal allergenis from a fungus taxonomically belonging to Alternaria or Cladosporium;(e) said dust mite allergen is from a dust mite taxonomically belongingto Dermatophagoides; (f) said animal allergen is from an animaltaxonomically belonging to Felis, Canis, or Equus; (g) said insectallergen is from an insect taxonomically belonging to Blatella,Periplaneta, or Hymenoptera; (h) said weed pollen allergen is from aweed taxonomically belonging to Urticales.
 22. The recombinant allergenof claim 21 wherein (a) said tree taxonomically belonging to Fagalesbelongs taxonomically to Betula, Alnus, Corylus, or Carpinus; (b) saidtree taxonomically belonging to Oleales belongs taxonomically to Olea;(c) said tree taxonomically belonging to Pineales belongs taxonomicallyto Cryptomeria; (d) said grass taxonomically belonging to Poales belongstaxonomically to Cynodon, Dactylis, Festuca, Holcus, Lolium, Phleum,Poa, Secale or Sorghum; (e) said herb taxonomically belonging toAasterales belongs taxonomically to Ambrosia, Artemicia, or Urticales;(f) said fungus taxonomically belonging to Alternaria belongstaxonomically to Alternata; (g) said fungus taxonomically belonging toCladosporium belongs taxonomically to herbarium; (h) said dust mitebelonging taxonomically belonging to Dermatophagoides belongstaxonomically to Farinae or Pteronyssinus; (i) said animal taxonomicallybelonging to Felis belongs taxonomically to Domesticus; (j) said animaltaxonomically belonging to Canis belongs taxonomically to familiaris;(k) said animal taxonomically belonging to Equus belongs taxonomicallyto caballus; (l) said insect taxonomically belonging to Blatella belongstaxonomically to Germanica; (m) said insect taxonomically belonging toPeriplaneta belongs taxonomically to americana; (n) said insecttaxonomically belonging to Hymenoptera belongs taxonomically toVespidae, Apidae; and (o) said weed taxonomically belonging to Urticalesbelongs taxonomically to Parietaria.
 23. The recombinant allergen ofclaim 20, wherein the recombinant allergen is derived from Ves v
 5. 24.The recombinant allergen of claim 23, wherein the allergen has one ormore amino acid substitutions selected from the group consisting of: i)Ala at position 72 of SEQ ID NO: 39; and ii) Ala at position 96 of SEQID NO:
 39. 25. The recombinant allergen of claim 20, wherein therecombinant allergen is derived from Bet v
 1. 26. The recombinantallergen of claim 25, wherein the allergen has one or more amino acidsubstitutions selected from the group consisting of: (i) Pro at position10 of SEQ ID NO: 37; (ii) Gly at position 25 of SEQ ID NO: 37; (iii) Thrat position 28 of SEQ ID NO: 37, Gln at position 32 of SEQ ID NO: 37;(iv) Ser at position 45 of SEQ ID NO: 37; (v) Ser at position 47 of SEQID NO: 37; (vi) Asn at position 55 of SEQ ID NO: 37; (vii) Ala atposition 77 of SEQ ID NO: 37; (viii) Gly at position 108 of SEQ ID NO:37; and (ix) Thr at position 28 of SEQ ID NO: 37, Gln at position 32 ofSEQ ID NO: 37, Ser at position 45 of SEQ ID NO: 37, and Gly at position108 of SEQ ID NO:
 37. 27. The recombinant allergen of claim 13, which isproduced by a method comprising: a) identifying amino acid residues insaid naturally occurring allergen which are conserved with more than 70%identity in all of the known homologous proteins within the taxonomicorder from which said naturally occurring allergen originates; b)defining at least one patch of said conserved amino acid residues whichpatch (i) is connected over at least 400 Å² of the surface of thethree-dimensional structure of said naturally occurring allergenmolecule; and (ii) comprises at least one B cell epitope, and wherein(iii) said conserved amino acids have a solvent accessibility of atleast 20%; and c) substituting at least one of said identified aminoacid residues in said defined at least one patch with a non-conservativeamino acid residue.
 28. A pharmaceutical composition comprising arecombinant allergen prepared according to claim 1 and apharmaceutically acceptable carrier.
 29. A pharmaceutical compositioncomprising the recombinant allergen according to claim 13 and apharmaceutically acceptable carrier.
 30. A method of generating animmune response in a subject, which method comprises administering tothe subject at least one recombinant allergen prepared according toclaim 1 or a pharmaceutically acceptable composition comprising said atleast one recombinant allergen.
 31. A method of generating an immuneresponse in a subject, which method comprises administering to thesubject at least one recombinant allergen according to claim 13 or apharmaceutically acceptable composition comprising said at least onerecombinant allergen.
 32. A method of vaccination or treatment of asubject, which method comprises administering to the subject at leastone recombinant allergen prepared according to claim 1 or apharmaceutically acceptable composition comprising said at least onerecombinant allergen.
 33. A method of vaccination or treatment of asubject, which method comprises administering to the subject at leastone recombinant allergen according to claim 13 or a pharmaceuticallyacceptable composition comprising said at least one recombinantallergen.
 34. A method of treatment, prevention or alleviation ofallergic reactions in a subject, which method comprises administering tothe subject at least one recombinant allergen prepared according toclaim 1 or a pharmaceutically acceptable composition comprising said atleast one recombinant allergen.
 35. A method of treatment, prevention oralleviation of allergic reactions in a subject, which method comprisesadministering to the subject at least one recombinant allergen accordingto claim 13 or a pharmaceutically acceptable composition comprising saidat least one recombinant allergen.